Opioid receptors (, d and k), are Gi/o-coupled, rhodopsin-like receptors. ? opioid receptor (KOPR) agonists may be useful as analgesics and antipruritic agents without abuse potential and respiratory depression associated with currently use opioid analgesics. However, prototypic selective KOPR agonists cause dysphoria or aversion, which limits their development. G protein-coupled receptors (GPCRs) signal via both G protein or arrestin to activate different downstream effectors. Biased agonists preferentially activate G protein- or arrestin-mediated signaling and thus may have advantages over balanced or unbiased agonists in that they may produce therapeutic effects with fewer side effects. However, translating in vitro ligand bias to in vivo pharmacology has been uncertain. Nalfurafine, the only selective KOPR agonist in clinical use, is prescribed in Japan for treatment of uremic pruritus, without causing dysphoria at therapeutic doses. In mice, we observed that nalfurafine caused conditioned place aversion (CPA) at doses higher than the effective doses for the antinociceptive and anti-scratch effects; however, the reverse was true for two other selective KOPR agonists, U50,488H and MOM-SalB. Similarly, U50,488H, but not nalfurafine, induced anhedonia. Thus, we established an animal model to understand the mechanisms underlying separation of anti-pruritic and analgesic effects from dysphoria / aversion of KOPR agonists. Importantly, we found in mouse brains U50,488H and MOM-SalB caused robust KOPR phosphorylation, but nalfurafine did not. Also,U50,488H, but not nalfurafine, enhanced phosphorylation of some proteins downstream of mTOR and the mTOR pathway may be involved in KOPR-mediated CPA. For Specific Aim 1, We will test the hypothesis that the ability of agonists to promote CPA is related to its ability cause KOPR phosphorylation by examining several structurally distinct KOPR agonists. KOPR phosphorylation will be detected with immunoblotting using our own antibodies that specifically recognize phosphorylated KOPR. For Specific Aim 2, we will examine the differences between U50,488H and nalfurafine in downstream phosphoproteomic changes in brain regions important in KOPR pharmacology. Furthermore, we will investigate the involvement of differentially regulated proteins / pathways in KOPR-mediated CPA and anhedonia. For Specific Aim 3, we will generate mutant mouse lines to examine the roles of agonist-promoted KOPR phosphorylation and GRK5 and GRK6 in KOPR pharmacology in vivo. KOPR-mediated antipruritic, antinociceptive, aversive and sedative effects and motor incoordination will be used as the in vivo pharmacological measures. Our ?from bedside to bench? approach, distinctly different from the commonly used ?from bench to bedside? strategy, allows us to circumvent the challenges of translating in vitro cell-based results to in vivo pharmacology. Taken together, the proposed studies will greatly advance our understanding of KOPR pharmacology at the molecular, cellular, and behavioral levels and signaling at a system level. Signaling pathways identified to be involved in KOPR-mediated aversion and anhedonia may shed light on mechanisms underlying aversion- and depression-like behaviors in general. In addition, it may lead to development of KOPR agonists that cause lower dysphoria and can be used as anti-itch medications and analgesics, which will contribute to solving the problems of the opioid abuse epidemic.